We have developed methods to site-specifically introduce fluorescent probes at polypeptide C-termini, at polypeptide hexahistidine tags, in DNA, and in RNA, and we are using fluorescence resonance energy transfer to define distances between pairs of fluorescent probes site-specifically introduced into transcription complexes. We are addressing the following issues (1) structural organization of subunits within RNA polymerase (2) extent of wrapping of DNA around RNA polymerase in initiation complexes (3) positions of the template and non-template strands of the transcription bubble in initiation complexes (4) path of the nascent RNA in elongation complexes (5) interactions with regulatory factors and (6) real-time, single-molecule analysis of initiation, elongation, and regulation.